This proposal is a resubmission of application R21MH103812-01, Long non-coding RNAs, learning and memory, which was reviewed in November 2013 by the MNG study section. Its focus is to establish a functional role for activity-dependent long non-coding RNAs in cortex. We are particularly interested in how non-coding RNAs may mediate epigenetic regulation of the gene expression underlying behavior and cognition. Eventually, we hope to examine how these enigmatic transcripts are involved in the fundamental molecular transactions underlying memory, and how they contribute to the development of fear- related psychiatric disorders. We have made an effort to address every issue that has been raised, and the grant has been substantially revised both to clarify the experimental approach and to include additional data. As a young investigator, I sincerely appreciate the thought and constructive criticism that went into the initial review, and I believe that the revised application is significantly stronger than the initial submission. Reviewer 1 indicated that, time should have been spent discussing what results would be expected from the ChIRP experiments and how they would be interpreted, and to propose to study how interaction of the lncRNA with the promoter of the protein coding genes leads to transcription regulation. We have now addressed this issue by including experiments utilizing ChIP assay for interrogation of the chromatin landscape surrounding TSS of protein coding gene targets, RIP for candidate lncRNA-chromatin modifying complex interactions and ChIRP for lncRNA-target protein coding gene promoter interactions. Moreover, we now describe how they will shed light on the role of lncRNAs in regulating gene expression associated with learning. Reviewer 1 also stated that, it would be instructive to know if and how the lncRNA acts as a decoy, a scaffold or a guide for regulating expression of the protein coding genes. We believe this is a great question and, although it is beyond the scope of the current exploratory R21 grant, it will definitely be subject of a future R01 application within the context of cell-type specific gene regulation supporting behavioral adaptation. Finally, it was unclear to reviewer 1 with whom the PI would collaborate and/or what services they will provide that will facilitate success of this project. Dr. Sha Sun in the Department of Developmental and Cell Biology at UCI is an expert in both lncRNAs and epigenetic programming (Sun et al., 2013) and has offered to serve as a collaborator on this project (letter of support attached) by providing strategic advice on the project as it progresses, and practical support in sharing expertise in the application of experimental validation of the novel findings to assist in the efficient and successful prosecution of this project. Both Reviewer 2 and 3 expressed concern that, the ASO technology may not be efficient at knocking down lncRNA, and that no other alternative approach was mentioned. It is important to note that we already have successfully manipulated Gomafu in primary cortical neurons in culture (Barry et al., 2013) and preliminary data, Aim 2). In the event that we can't reduce Gomafu or other lncRNAs in vivo using ASO, we propose to use CRISPR/Cas9 technology to knockdown these lncRNA. However, we also acknowledge that this approach carries some degree of risk, due to the permanent nature of the knockout, which may lead to compensatory effects on the expression of genes that were under inhibitory control by the target lncRNA. Finally, we have a Gomafu lncRNA overexpression construct that will also be tested in Aim 3. Reviewer 3 indicated that, it would also be important to determine whether there is indeed a functional relationship between lncRNA and its protein-coding targets. E.g. can the effect of manipulating lncRNA be counter-acted by the manipulation of protein-coding target? We completely agree with this reviewer; however, due to time limitations for the R21 project, these experiments will be included in a future R01 designed to explore in greater detail the functional relationship between lncRNA-mediated epigenetic regulation of gene expression, once a role for candidate lncRNAs in regulating learning and memory have been firmly established.